This basic research proposal in mice dissects the neural circuitry and receptors that underlie therapeutic vs side effects of the sleep medicines used to treat Veterans, using a state-of-the-art gene editing approach called clustered regularly interspaced short palindromic repeats (CRISPR). The support of this CDA2 award would allow the applicant to be trained in in vivo reverse microdialysis and in vitro electrophysiology to allow him to comprehensively validate his genetic manipulations and dissection of neurocircuits, and would allow him to become a leader in pre-clinical sleep research within the VA. Disturbed sleep occurs in neuro-psychiatric illnesses such as insomnia, sleep apnea, post-traumatic stress disorder and traumatic brain injury. United States Veterans have more than double the amount of sleep disturbance compared to the rest of the population. As a result, sleep medicines like zolpidem (Ambien) and eszopiclone (Lunesta) are prescribed widely to Veterans. From 2005 to 2014, VA prescriptions of zolpidem increased nearly 7 times, and VA prescriptions of eszopiclone increased over 100 times for men and over 50 times for women Veterans. However, these medications do not promote a natural sleep and have side effects. Thus, a better understanding of their mechanism of action is needed to develop better treatments. Delta waves are slow brain rhythms at the speed of 0.5 to 4 waves per second, and large amounts of these waves are a defining feature of `deep' NREM sleep. Delta waves are linked to the restorative aspects of deep sleep (mood regulation, synaptic homeostasis, cellular energy regulation and clearance of toxic proteins). Problematically, zolpidem and eszopiclone induce `light' sleep and drastically reduce NREM delta waves. So perhaps unsurprisingly, these drugs are linked to suicide risk and cognitive problems. Delta waves are recorded from the cerebral cortex by electroencephalography, but they are generated deep within the brain's core structure, the thalamus. Excitatory ?Thalamocortical (TC)? neurons form the connections from the thalamus to the cortex, and they act as delta wave pacemakers. But they require an inhibitory drive to perform this function. This inhibitory drive is provided by the neurotransmitter GABA, which comes from an outer shell- like part of the thalamus called the thalamic reticular nucleus (TRN). Recent discoveries have shown that a stimulated TRN promotes delta waves. TRN neurons themselves, receive GABA from wake active neurons in the basal forebrain and lateral hypothalamus. With this in mind, we will test a hypothesis that GABAergic inhibition onto TRN regulates delta waves via GABAergic inhibition onto TC neurons. This will be the 1st study in this topic that dissects molecular, cellular, and brain-region specific mechanisms simultaneously in vivo. ?3 subunits are a major structural component of the type of GABAA receptors that are native to TRN. In Specific Aim (SA) 1 we use CRISPR-Cas9 to locally ablate ?3 subunits within a subset of TRN neurons that are defined by the presence of a calcium-binding protein called parvalbumin (PV). Our preliminary data shows that disrupting this GABA transmission increases NREM delta waves and promotes NREM in vivo; and reduces spontaneous inhibitory post synaptic currents (sIPSC) in vitro. To add rigor and reproducibility, we use an alternative mouse genetic approach to overexpress ?3 subunits in PV+ TRN neurons. Training will enable the in vitro work. In SA2 we use CRISPR-Cas9 to locally ablate ?1 subunits, which form the type of GABAA receptors that are native to TC neurons. Here we focus on the paraventricular thalamus, which is involved in stress-induced arousal. We will also overexpress ?1 in the TC neurons. In vitro data will be collected during the training. In SA3 Dr Uygun will train to use in vivo reverse microdialysis to locally administer eszolpiclone and zolpidem to TRN and TC neurons. This will examine the delta suppressing component of sleep medicines. This work will guide the development of next-generation GABAergic sleep medicines, leading to improved Veteran patient care with lower suicide risk and better mood and cognitive performance.